Data from several animal models, as well as anecdotal evidence in humans, suggest that antibodies (Abs) to the protective antigen (PA) of anthrax are protective if administered in a timely manner. Unfortunately, a large and stable source of such Abs is not available to stockpile for use in case of a bioterrorist attack with Bacillus anthracis; however, cell lines making anti-PA monoclonal antibodies (mAbs) would provide a ready source for such reagents. While rodent anti-PA mAbs have been described, none has shown protection in animal models against fully virulent strains, although one did lead to a significant delay in guinea pigs in time to death. These negative results may be attributable to the use of mAbs specific for epitopes that do not neutralize the toxins and/or to the need to use a combination of two or more anti-toxin mAbs that would act synergistically to protect against the lethal effects of anthrax toxins. It is also possible that the mouse B cell repertoire against PA does not sustain a strong response against epitopes that are involved in toxin neutralization. Production and use of human monoclonal antitoxins would have many advantages: they are, by definition, completely human and are therefore usable without engineering as therapeutic reagents in humans. Moreover, they are representative of the protective human polyclonal Ab response against anthrax and would therefore provide information about the epitopes and Ab mimotopes that could be used to develop molecular vaccines, which would be more efficacious than those currently in use or in development. For these reasons, we propose experiments to produce and characterize human mAbs against PA of anthrax. Thus, in Aim 1 we propose to produce heterohybridomas making human anti-PA mAbs. These will be produced from the peripheral blood of human volunteers who are recipients of an experimental vaccine consisting of recombinant PA (rPA). Cells from vaccinees will be obtained ~1 week after the final immunization, transformed with Epstein-Barr virus, cultured and screened for anti-PA production. Cells in Ab-positive wells will be fused with a heteromyeloma line, repeatedly screened for reactivity, and cloned at limiting dilution until monoclonality is achieved. In Aim 2, the various mAbs made will be studied to determine the epitopes, which they recognize, and how specificity, affinity and IgG subtype correlate with protective activity. Protective functions will be assessed in vitro by measuring the ability of individual mAbs or combinations of mAbs to block the lethal effects of anthrax toxin.